Millions of people suffer from chronic pain, which is a major health problem. Chronic orofacial pain is highly prevalent in the US. The most common persistent orofacial pain condition, temporomandibular joint disorders (TMJD), affects the musculoskeletal and joint tissues and is heterogeneous in origin. The current treatment for chronic pain conditions is unsatisfactory and there is an urgent need for searching and developing alternative and effective chronic pain therapy. Recently, bone marrow stromal cells (BMSC) have generated considerable interest as a candidate for cell-based therapy. Interestingly, BMSC appear to have potential to treat chronic pain conditions. In rat models of tissue or nerve injury with long-lasting pain hypersensitivity, intravenous infusion of rat BMSC produced long-term attenuation of orofacial hyperalgesia/allodynia (antihyperalgesia) and this effect was attenuated by the opioid receptor antagonist, suggesting the involvement of endogenous opioids. However, the mechanisms of the effect of BMSC on persistent pain remain elusive. Evidence indicates that the majority of the intravenously infused BMSC are trapped in the lungs and that the infused cells only stay in the system for a matter of days to a few weeks. Studies suggest that the infused BMSC produce their therapeutic effects through secretion of chemical mediators that interact with the body's immune system. We hypothesize that BMSC produce pain-relieving, or antinociceptive effect through their immune interactions and subsequent activation of the endogenous opioid system. We will test this hypothesis by a combination of approaches involving cell cultures, Reverse Transcription-quantitative real time Polymerase Chain Reaction, immunohistochemistry, Western blot, Enzyme-linked immunosorbent assay, fluorescence activated cell sorting, RNA interference (RNAi), transgenic mice and behavioral pharmacology in three Specific Aims. We will continue to use a rodent model of the masseter muscle tendon injury, which mimics prolonged orofacial nociception of myogenic origin. We will focus on using female animals since women exhibit a higher prevalence of TMJ disorders than men. Aim 1 will examine the effect of BMSC on pain and neuronal activity in female animals and test the hypothesis that BMSC engage brain endogenous opioids and regulate N-methyl-D-aspartate receptors. Aim 2 will test the hypothesis that the monocyte/macrophage population of immune cells are involved in mediating the BMSC-produced pain relief. Aim 3 will test the hypothesis that monocyte-derived chemokines are critical in the BMSC-produced upregulation of opioid receptors and attenuation of persistent pain. Findings will provide novel cellular mechanisms for BMSC-induced pain relief and help to develop an approach to effectively engage the endogenous pain modulation for the treatment of chronic pain and facilitate translating it into clinical settings.